Ink composition for bioprinting and hydrogel formed from the same

ABSTRACT

Provided are an ink composition for bioprinting and a hydrogel formed therefrom, wherein the ink composition: a monomer or macromer having a photocurable functional group; and acrylic hyperbranched polyglycerol (AHPG).

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Korean Patent Application No.10-2019-0028270, filed on Mar. 12, 2019, in the Korean IntellectualProperty Office, the disclosure of which is incorporated herein in itsentirety by reference.

BACKGROUND 1. Field

One or more embodiments relate to an ink composition for bioprinting.

2. Description of Related Art

There is a growing interest in engineering miniaturized softbiomaterials for such applications as complex tissue constructs and drugdelivery systems, with the recent advancement in microfabricationtechnology, such as digital light processing (DLP), stereolithography(SLA), and extrusion-based dispensing. Often collectively termed as“bioprinting” or “biofabrication”, this technology is deemed especiallyattractive for biomedical engineering applications, because, as theconcept of personalized medicine being hailed as the future paradigm ofmedicine. It is becoming ever more critical to rapidly produce tissueconstructs and drug delivery systems with desired architecture andresolution, while controlling the biological functions of engineeredtissues and drug release kinetics.

However, the microfabrication of polymer-based materials has largelybeen focused on conventional thermoplastic materials, such aspoly(lactic acid) (PLA) and acrylonitrile-butadiene-styrene (ABS) viahigh-temperature melt extrusion used primarily for structural support,which is not suitable for encapsulating sensitive biological entitiesfor tissue engineering and drug delivery applications.

With the continued maturation of biofabrication technology, the focus isnow shifting towards developing “bioinks.” Bioinks can not only beconverted to a solid structure upon printing in a timely manner via asuitable crosslinking scheme, but also the resulting structure providesprotection and suitable microenvironment for the encapsulating species.For this reason, the biofabrication technology is actively recruited toengineer various hydrogel-based structures. Hydrogels are widely used asscaffolds to support cells and tissues for various applications inregenerative medicine. Their mechanical properties can be tuned toprovide regulatory physical signals to optimize various cellularfunctions, while providing protection against harmful externalenvironment. Furthermore, the hydrogels can be engineered to presentcell-recognition molecules (e.g. ECM proteins, cell adhesion peptides)to enhance their affinity towards the polymeric network for attachment.

Earlier efforts of hydrogel biofabrication mostly relied on naturalpolymers that undergo rapid crosslinking to form hydrogels by physicalcrosslinking, allowing for fabrication via conventional extrusion-baseddispensing systems. For example, alginate hydrogels could be printed byusing the extruded alginate solution via crosslinking using calciumions. Agarose hydrogels can be fabricated by lowering the temperature toinduce the physical crosslinking of the extruded solution which is keptat liquid state at elevated temperatures prior to printing.

Although this type of fabrication is generally straightforward due toits simple crosslinking mechanisms, the same cannot be said forcontrolling their material properties. This is because, when anychemical modification is made to natural polymers to control thecharacteristics of hydrogel, the fluid mechanics and/or the crosslinkingefficiency that are crucial elements for the microfabrication processcould be inadvertently changed. More recently, due to the widespread useof various photocrosslinked hydrogels in biomedical applications, lightcuring-based printing systems such as DLP and SLA are increasinglyemployed to generate micrometer-scale hydrogel constructs made fromcommercially available monomers and macromers, such as acrylamide,methyl methacrylate, or sodium acrylate, to known polymers conjugatedwith photolabile functional groups, such as poly(ethylene glycol)diacrylate (PEGDA), methacrylic alginate, or methacrylic gelatin. Withthe relatively simple modification to impart photocrosslinkingcapability, it is more applicable to a variety of polymers thanextrusion-based systems.

SUMMARY

One aspect is to provide a novel ink composition for bioprinting.

Another aspect is to provide a crosslinked hydrogel formed byirradiating light to the ink composition for bioprinting.

Additional aspects will be set forth in part in the description whichfollows and, in part, will be apparent from the description, or may belearned by practice of the presented embodiments of the disclosure.

One aspect provides an ink composition for bioprinting including

a monomer or macromer having a photocurable functional group, and

acrylic hyperbranched ployglycerol (AHPG)

Another aspect provides a crosslinked hydrogel formed by irradiatinglight to a ink composition for bioprinting, and

the ink composition including: a monomer or macromer having aphotocurable functional group; and AHPG.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of certainembodiments of the disclosure will be more apparent from the followingdescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1-1 shows synthesis of acrylic hyperbranched polyglycerol (AHPG)crosslinker, FIG. 1-2 shows the control of crosslinking density ofhydrogels depending on the degree of substitution (DS) of acrylate ofAHPG, and FIGS. 1-3 to 1-6 show the change in shear viscosity of eachmaterial with shear rate;

FIGS. 2-1˜2-9 show the elastic modulus (E) ofpoly(ethylenglycol)methacrylate (PEGMA) hydrogel and polyacrylamide(PAAm) hydrogel, each crosslinked with AHPG having variousconcentrations, molecular weights (2000 (2K), 5000 (5K), 15000 (15K) gmol⁻¹) and acrylate DS;

FIGS. 3-1˜3-5 show elastic modulus (E) of hydrogels having various AHPGconcentrations and acrylate DS;

FIGS. 4-1˜4-8 show the cumulative drug release profile and the like of adrug encapsulated in a hydrogel;

FIG. 5-1 shows images of cells encapsulated in MGel-AHPG5K hydrogelshaving various acrylate DS (AHPG concentration ratio (ΦAHPG)=0.1), andFIGS. 5-2˜5-7 show diagrams of viability and proliferation rates;

FIGS. 6-1˜6-4 show diagrams schematically showing the microfabricationof AHPG-crosslinked hydrogel via DLP projection printing;

FIG. 7-1 shows, ¹³C-NMR spectrum, FIG. 7-2 shows ¹H-NMR spectrum, andFIG. 7-3 shows the integrated value of the peaks corresponding tovarious molecular weights, in relation to HPG; and

FIG. 8 shows the ¹H-NMR spectrum of AHPG.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings, wherein like referencenumerals refer to like elements throughout. In this regard, the presentembodiments may have different forms and should not be construed asbeing limited to the descriptions set forth herein. Accordingly, theembodiments are merely described below, by referring to the figures, toexplain aspects of the present description. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items. Expressions such as “at least one of,” whenpreceding a list of elements, modify the entire list of elements and donot modify the individual elements of the list.

An ink composition for bioprinting according to one aspect includes: amonomer or macromer having a photocurable functional group; and acrylichyperbranched ployglycerol (AHPG).

According to one embodiment, the concentration of AHPG in the inkcomposition for bioprinting may be from about 0.1% (w/v) to about 5%(w/v) based on the total volume of the ink composition. Theconcentration of AHPG may be from about 3% (w/v) to about 10% (w/v).

According to one embodiment, the concentration of the monomer ormacromer having a photocurable functional group may be from about 1%(w/v) to about 30% (w/v) based on the total volume of the inkcomposition. The concentration of the monomer or macromer having aphotocurable functional group may be from about 4% (w/v) to about 20%(w/v), for example, from about 4% (w/v) to about 15% (w/v), or fromabout 10% (w/v) to about 20% (w/v).

According to one embodiment, the concentration of the monomer ormacromer having a photocurable functional group; and AHPG may be, basedon the total volume, from about 5% (w/v) to about 30% (w/v), forexample, from about 8% (w/v) to about 25% (w/v), or from about 5% (w/v)to 15% (w/v).

According to one embodiment, the molecular weight of AHPG may be fromabout 2000 Da to about 15000 Da, for example, about 2000 Da to about10000 Da, or about 5000 Da to about 15000 Da.

According to one embodiment, a hyperbranched hydrogel glycerol moiety inthe AHPG for the ink composition for bioprinting, may be prepared byreacting polyhydric alcohol with glycidol and may have a molecularweight of about 1700 Da to about 15000 Da, for example, about 1700 Da toabout 10000 Da, or about 4000 Da to about 15000 Da. The polyhydricalcohol refers to a compound in which two or more OH groups are bondedto an alkyl group, and the polyhydric alcohol may be, for example, atrihydric alcohol or a tetrahydric alcohol. Non-limiting examples of thepolyhydric alcohol include trimethylol ethane, trimethylol propane,pentaerythritol, di(trimethylol propane), and the like.

According to one embodiment, AHPG of the ink composition for bioprintingis prepared by reacting the OH end of the hyperbranched polyglycerol(HPG), prepared by reacting glycidol with polyhydric alcohol, with acompound having an acrylic group. In AHPG, 5 to 80% of the hyperbranchedployglycerol (OH) groups of HPG are substituted with an acrylic group.That is, the degree of substitution (DS) of the OH group of HPG by theacrylic group may be from about 5% to about 80%. For example, the DS maybe from about 10% to about 70%.

According to one embodiment, the acrylic group may be acrylate ormethacrylate.

The monomer having the photocurable functional group is not particularlylimited, and according to one embodiment, the monomer having thephotocurable functional group may be water-soluble, for example,acrylamide.

The molecular weight of the macromer having the photocurable functionalgroup may be from about 400 Da to about 50000 Da.

The macromer having the photocurable functional group is notparticularly limited and may be, for example, poly(ethylene glycol)methacrylate (PEGMA). The molecular weight of PEGMA may be from about400 Da to about 5000 Da, for example, about 700 Da to about 2000 Da, orabout 1000 Da to about 4000 Da. When the molecular weight of PEGMA isless than 400 Da, the solubility thereof with respect to aqueoussolvents is significantly decreased, and when the molecular weight ofPEGMA is greater than 5000 Da, the molecular fluidity is poor and thenumber of functional groups is reduced, thereby making gel formationdifficult.

According to one embodiment, the water-soluble macromer having aphotocurable functional group may be methacrylic gelatin (MGel).Methacrylic gelatin is gelatin bonded with a number of methacrylicgroups, and a specific production method therefor is known, and thuswill be omitted herein.

According to one embodiment, the ink composition may further include anaqueous solvent, and the aqueous solvent may be, for example, phosphatebuffered saline (PBS) of pH 7.0 to pH 8.0, for example, 7.4.

According to one embodiment, the ink composition for bioprinting has ashear-thinning property. Therefore, when the ink composition is used asthe ink of the 3D printer, since the ejection from the nozzle is smooth,the clogging phenomenon is significantly reduced, and the viscosity ofthe ejected ink returns to the viscosity before the ejection.

According to one embodiment, the ink composition may further include aphotoinitiator, and the photoinitiator is not particularly limited, andaccording to one embodiment, may be2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone.

According to another aspect, the hydrogel may be prepared by irradiatinglight to an ink composition for bioprinting to form crosslinking bonds,wherein the ink composition includes a monomer or macromer having aphotocurable functional group and AHPG.

According to one embodiment, the ink composition for bioprinting mayfurther include an aqueous solvent, and the aqueous solvent may bephosphate buffered saline (PBS).

According to one embodiment, the light may be ultraviolet (UV), and thewavelength of the UV may be from about 200 nm to about 450 nm, and theirradiation time may be from about 10 seconds to about 5 minutes. Whenthe wavelength of the UV is less than 200 nm, the energy is too strong,and when the wavelength of the UV is more than 450 nm, the energy is toosmall to cause crosslinking. When the irradiation time is less than 10seconds, crosslinking is not sufficiently achieved, and when it is morethan 5 minutes, the irradiation time adversely affects drugs, cells,etc., which may be additionally added.

According to one embodiment, when the ink composition for bioprintingfurther includes a drug, the hydrogel may be used in a drug deliverysystem.

According to one embodiment, when the ink composition for bioprintingfurther includes a cell, the hydrogel may be used as a scaffold for cellculturing for tissue engineering for biomedical applications.

A hydrogel system based on a hyperbranched polyglycerol (HPG)crosslinker was developed in order to control the mechanical propertiesof hydrogels made from various monomeric systems in a more refinedmanner. HPG possesses several characteristics that are highly suited forbiomedical applications, such as hydrophilicity, biocompatibility, and asimple synthetic route. In addition, the molecular weight and the degreeof branching could also be easily controlled during the synthesis totune the viscoelastic properties.

With numerous hydroxyl groups on a HPG molecule, it is also possible tocontrol the degree of substitution (DS) of reactive functional groupsrequired for crosslinking reaction. Here, a varying number of acrylatewas bonded by a chemical reaction of hydroxyl groups on HPG to developacrylic HPG (AHPG) to impart photocrosslinking capability. The effectsof DS of acrylate, which in effect controls the hydrophilic/hydrophobicbalance, and molecular weight of AHPG on the mechanical properties ofresulting hydrogels were explored at various types of monomers tovalidate the necessity of tuning those physical properties of polymericcrosslinker for a particular monomer for optimal physical interactionthat ultimately affect the hydrogel mechanics.

With this mechanical tunability as well as photocrosslinking capability,a DLP projection printing system was utilized to generateAHPG-crosslinked microgels (micrometer-sized hydrogel) with complexarchitecture and varying mechanical properties to demonstrate theAHPG-crosslinked hydrogel as versatile bioink material for bioprintingapplications.

Synthesis of Photocrosslinkable AHPG

Unlike traditional dendrimers with precise molecular weight andwell-defined shape, which are synthesized via sequential generation ofbranches (identified by the number of “generations”), hyperbranchedpolymers are developed by one-pot polymerization of monomers thatgenerate random branching points, offering a more scalable and practicalroute for creating branched, multivalent macromolecules. HPG hasgarnered significant interest in the area of biomedical engineering, fortheir hydrophilicity and biocompatibility as well as their facilefabrication scheme. That is, HPG is generally considered a branchedpoly(ethylene glycol) due to its polyoxyether backbone. In addition,their multivalent hydroxyl groups provide an avenue for chemicalmodification to impart desired functionalities. Herein, HPGs havingvarious molecular weights (M_(w)) were synthesized and their hydroxylgroups were bonded with photolabile acrylic groups. The acrylic HPG wasthen used as a multifunctional crosslinker to fabricate hydrogels withvarying mechanical properties (FIG. 1-2). For conventional linearpolymers, the number of functional groups are often limited, mostly tothe end groups. On the other hand, branched polymers such as HPG containnumerous functional groups that could be converted to reactivefunctional groups in a given molecular weight. Moreover, the moleculardynamics of hyperbranched polymers would be less affected by theaddition of the functional groups than linear polymers at the samemolecular weight due to significantly less chain entanglement That is,the length of each chain on hyperbranched polymer is shorter than thatof a linear polymer at the same M_(w).

HPG was synthesized via anionic ring opening polymerization of glycidolas the monomer (FIG. 1-1). The M_(w) could be controlled efficiently bythe ratio of trimethylolpropane (TMP), to monomer. HPG with threedifferent M_(w) were synthesized; 1751, 4553, and 14 418 g mol⁻¹ asdetermined from ³C-NMR spectra, or 2230, 4350, and 14 300 as determinedfrom gel permeation chromatography (GPC), which showed they are in goodagreement. Herein, they are denoted as 2K, 5K, and 15K, respectively,for convenience. In addition, the ratio of TMP to base activator (i.e.,NaH) was kept constant in order to keep the degree of branchingconstant, and only control the length of branches. Controlling thelength of the branches of HPG was expected to modulate the interactionbetween the monomers (or macromers), resulting in various mechanicalproperties of the resulting hydrogels.

Controlling the M_(w) of hyperbranched polymers such as HPG, as comparedwith linear polymers at the same range, was also expected to cause muchsmaller change in viscoelastic properties, which is an important factorfor bioprinting applications where it is critical to control the fluidproperties of ink materials. To evaluate the change in viscoelasticproperties of HPG with varying M_(w), the viscosity versus shear ratewas measured. As a control, linear poly(ethylene glycol) (PEG) atsimilar M_(w) were also investigated. With increasing shear rate, thedistinctive shear-thinning behavior was shown for all HPG, in whichthere was a sharp decrease in shear viscosity when the shear rate wasincreased from 0.03 to 0.6 s⁻¹ for all HPG at the same rate (FIG. 1-3).In addition, the decrease occurred at the same region of shear rate. Forlinear PEG, on the other hand, the shear viscosity was generally higherat all shear rate, and the decrease with shear rate was more gradual(FIG. 1-4). Also, the viscosity at higher shear rate was more variedwith M_(w) than HPG. This result signified that the chain relaxationmore readily occurred and the viscosity was less affected by the M_(w)for HPG than the respective linear polymer, allowing for more extensivecontrol of M_(w) and concentrations of HPG without affecting theviscoelastic behavior.

FIG. 1-1 shows synthesis of acrylic hyperbranched polyglycerol (AHPG)crosslinker, FIG. 1-2 shows the control of crosslinking density ofhydrogels depending on the degree of substitution (DS) of acrylate ofAHPG, and FIGS. 1-3 to 1-6 show the change in shear viscosity of eachmaterial with shear rate.

FIG. 1-1 schematic diagram illustrating a process in which HPG wassynthesized via anionic ring opening polymerization of glycidol as themonomer, and AHPG was obtained by nucleophilic reaction between hydroxylgroups of HPG and acryloyl chloride.

FIG. 1-2 shows a schematic view illustrating that the crosslinkingdensity of hydrogels can be controlled by changing the degree ofsubstitution (DS) of acrylate of AHPG.

FIG. 1-3 shows the measurements of the shear viscosity change withrespect to the shear rate for HPG having a variety of M_(w), and FIG.1-4 shows the measurements of the shear viscosity change with respect tothe shear rate for PEG with a variety of M_(w). In FIGS. 1-3 and 1-4,the abscissa is the shear rate, and the ordinate is the shear viscosity.

FIG. 1-5 shows a graph of shear viscosity with respect to a shear rate,in relation to 20% (w/v) PEGMA-3% AHPG5K at varying acrylate DS, andFIG. 1-6 shows a graph of shear viscosity with respect to a shear rate,in relation to 20% (w/v) PEGMA-AHPG5K (DS3) with varying concentration.In FIGS. 1-5 and 1-6, the abscissa is the shear rate, and the ordinateis the shear viscosity.

Ultimately, HPG was bonded with varying number of acrylate to developacrylic HPG (‘AHPG’) via nucleophilic substitution of the hydroxylgroups (FIG. 1-1). At a given M_(w) of HPG, the DS of acrylate wascontrolled; 10, 30, 50, and 70% of the hydroxyl groups, which aredenoted as DS1, DS2, DS3, and DS4, respectively, for convenience (FIG.2-1˜2-9). It was hypothesized that the mechanical properties of thehydrogels crosslinked with AHPG could be controlled with the acrylateDS, even without changing the concentration. This would also ensure thatthe viscoelastic properties of various precursor solutions could remainlargely constant while trying to control the mechanical properties ofresulting hydrogels with either M_(w) of acrylate DS.

Mechanical Properties of AHPG-Crosslinked Hydrogels

Hydrogels crosslinked with AHPG were fabricated via photocrosslinkingand their mechanical stiffness was measured to assess the effect ofphysical properties of AHPG, M_(w) and acrylate DS, on the crosslinkingreaction with different monomers, and subsequently the hydrogelmechanics. With the unconventional molecular architecture (i.e.,hyperbranched), varying hydrophilic-hydrophobic balance arising fromacrylate DS (i.e., hydrophilic polyoxyethylene backbone and hydrophobicacrylate), it was hypothesized that the AHPG would have significantinfluence over different types of monomers. Therefore, hydrogels madefrom three different types of widely used monomers were investigated;small molecule (e.g., acrylamide), macromer (e.g., poly(ethylene glycol)methacrylate (PEGMA)), and protein (e.g., methacrylic gelatin (MGel)).The hydrogel stiffness was evaluated by calculating compressive elasticmoduli.

FIGS. 2-1 to 2-6 show diagrams of elastic moduli (E) of PEGMA hydrogelscrosslinked with AHPG having varying concentrations and acrylate DS, andFIGS. 2-7 to 2-9 show diagrams of elastic moduli (E) of PAAm hydrogelcrosslinked with AHPG having varying concentrations and acrylate DS.

The M_(w) of AHPG are (in FIGS. 2-1, 2-4, and 2-7) 2K, (in FIGS. 2-2,2-5, and 2-8) 5K, and (in FIGS. 2-3, 2-6, and 2-9) 15K. Theconcentrations of PEGMA for FIGS. 2-1 to 2-3 are 10% (w/v), and theconcentrations of PEGMA for FIGS. 2-4 to 2-6 are 20% (w/v). Theconcentrations of PAAm for FIGS. 2-7 to 2-9 are 20% (w/v). In FIGS. 2-1to 2-9, the abscissa represents the DS of acrylate, and the ordinaterepresents the modulus.

Macromer: Poly(Ethylene Glycol) Monoacrylate (PEGMA)

First, PEGMA hydrogels crosslinked with AHPG were developed and theirelastic moduli were obtained (FIGS. 2-1 and 2-3). The concentration ofPEGMA was controlled at 10% (w/v), while that of AHPG was controlledfrom 0.5 to 3% (w/v) for 10% (w/v) PEGMA, or from 1% (w/v) to 5% (w/v)for 20% (w/v) PEGMA. At low M_(w) AHPG at 2K, hydrogels did not formuntil acrylate DS of AHPG was increased to DS3, and the elastic moduliwere larger at DS4 than DS3 (FIG. 2-1). On the other hand, at AHPG5K,the elastic moduli of hydrogels were generally larger and the hydrogelsformed at lower concentration and acrylate DS than AHPG2K (FIG. 2-2).This suggested that at the given concentration, significant branchlength was required to interact with the macromer chains leading toenhanced crosslinking efficiency. It should be emphasized that comparedto linear polymer at the same M_(w), hyperbranched polymers are morecompact and thus less likely to physically interact with other polymerchains. Interestingly, there was a decrease in moduli when the acrylateDS was increased beyond a certain value; DS4 for 3% (w/v) and DS3 for 1%(w/v). With increasing hydrophobicity at higher acrylate DS, diminishedphysical association between hydrophilic macromer may have reduced theextent of crosslinking reaction. When the M_(w) of AHPG was furtherincreased to 15K, there was an increase in moduli up to DS2, especiallyat 3% AHPG (FIG. 2-3). However, the elastic moduli decreased moresharply at higher acrylate DS, suggesting that the increasedhydrophobicity with AHPG with higher acrylate DS likely causedinsufficient physical association between PEGMA and AHPG, more so athigher M_(w) with decreased chain conformational flexibility andmobility, further diminishing the extent of crosslinking. Thisexplanation was further corroborated by the increased opaqueness andmicroscopic heterogeneity shown for hydrogels crosslinked with AHPG athigher acrylate DS and concentration, which suggested there wassignificant phase separation within the hydrogel network. These resultsnot only demonstrate the control of mechanical properties of hydrogelswith AHPG, but also highlight the importance of considering the physicalproperties of the AHPG crosslinkers, namely, the chain length andhydrophilic-hydrophobic balance, when choosing the macromer conditionfor preparing hydrogels.

When the concentration of PEGMA was increased to 20% (w/v), the range ofelastic moduli was much greater, and the hydrogels formed even at loweracrylate DS and concentrations of AHPG than the hydrogels at 10% (w/v)PEGMA (FIGS. 2-4 and 2-6). With AHPG2K, the hydrogel formed at allacrylate DS and the elastic moduli increased with acrylate DS at allconcentrations, from 1 to 5% (FIG. 2-4). In addition, there was muchgreater increase in moduli at higher M_(w) of AHPG, 5K and 15K, by theacrylate DS and concentration than those at 10% PEGMA hydrogels (FIGS.2-5 and 2-6). These results demonstrated that the branched polymericchains of AHPG could extensively incorporate the increased amount ofmacromers to form the hydrogel network. Similar to 10% (w/v) PEGMAhydrogels, the elastic moduli decreased above a critical acrylate DS athigher concentration and M_(w) of AHPG, further confirming the role ofdiminished chain movement and increased hydrophobicity on the extent ofcrosslinking between PEGMA and AHPG.

The viscosity versus shear rate curves for the PEGMA-AHPG hydrogelprecursor solutions were measured to assess the effects of M_(w) andconcentration of AHPG on their fluid behavior (FIGS. 1-5 and 1-6). Thechanges in viscosity in response to varying shear rate were notsignificantly affected, and the well defined shear-thinning behavior wasdemonstrated, regardless of the M_(w) and acrylate DS of AHPG. Thisresult further established that the use of hyperbranched polymericcrosslinker could control the mechanical properties of hydrogels withconsistent fluid properties with shear-thinning behavior, validatingAHPG as an effective ink material for microfabrication.

Small Molecular Monomer: Acrylamide

Polyacrylamide (PAAm) hydrogels, prepared by copolymerization betweenacrylamide monomers and crosslinkers, have long been utilized in variousbiomedical applications, such as gel electrophoresis, medical implants,and drug delivery systems. Thus, AHPG was further investigated as acrosslinker for acrylamide monomers to fabricate PAAm hydrogels, andtheir mechanical properties were evaluated (FIGS. 2-7 and 2-9). ForAHPG2K and AHPG5K, the range of elastic moduli controlled by the AHPGconcentration was similar, and the minimum acrylate DS for hydrogelfabrication was the same at DS2 (FIGS. 2-7 and 2-8). This resultsuggested the small molecular monomers such as acrylamide having greatermobility than larger macromers could more readily interact with AHPGcrosslinkers, such that the change in M_(w) of AHPG within this rangedid not have a significant effect on the crosslinking.

When the M_(w) of AHPG was further increased to 15K, there was a drasticdecrease in mechanical properties at all concentrations and acrylate DSby more than 50%, up to 80% (FIG. 2-9). This significant drop may havebeen due to the larger size of AHPG disrupting the PAAm chain growth,the process that was not necessary for the macromers such as PEGMA,which already possessed polymeric chain structure. From the standpointof AHPG, it is plausible that the limited conformational andtranslational mobility of longer chains of AHPG favored self-associationvia chain entanglement rather than interaction with growing PAAm chains.This was further strengthened by the fact that the elastic moduli werethe highest at DS1 and decreased with acrylate DS, which likely resultedfrom the separation of AHPG with higher acrylate DS from growing PAAmchains due to increased hydrophobicity.

Multivalent Macromer: Methacrylic Gelatin

FIGS. 3-1 and 3-2 show diagrams of elastic moduli (E) of MGel hydrogelhaving acrylate DS at 4% (w/v) MGel, and at varying concentrations ofAHPG from 0.1% (w/v) to 1% (w/v), and FIGS. 3-3 and 3-4 show diagrams ofelastic moduli (E) of MGel hydrogel having acrylate DS when the totalpolymer concentration was kept constant at 10% (w/v), while varying thefraction of AHPG (ΦAHPG). The M_(w) of AHPG is 2K in FIG. 3-3, 5K inFIGS. 3-1 and 3-4, and 15K in FIGS. 3-2 and 3-5.

Polymers having multiple functional groups on the backbone can be bondedwith crosslinkable functional groups in a varying degree ofsubstitution. This type of “multivalent” polymers can either formhydrogel by themselves above a critical concentration, or be crosslinkedor hybridized with another crosslinker to form hydrogels at a lowerconcentration. To further evaluate the crosslinking ability of AHPG,photocrosslinkable gelatin which contains multiple methacrylic groups(MGel) as a model macromer was crosslinked with AHPG to develophydrogels, and their mechanical properties were similarly evaluated(FIG. 5-1˜5-7). Photocrosslinkable MGel-based hydrogels are widely usedas scaffold materials for tissue engineering applications. With theamphiphilic nature of gelatin as a protein, it also suggested that theacrylate DS of AHPG could also play a significant role in controllingthe hydrogel mechanics.

The average molecular weight of MGel may range from about 400 Da toabout 50000 Da, for example, 3000 Da to 50000 Da, for example, 10000 Dato 40000 Da.

First, the concentration of MGel was kept at 4% (w/v), which is belowthe critical concentration for hydrogel formation by itself (5% (w/v)),and the concentration of AHPG was controlled from 0.5% (w/v) to 3% (w/v)in order to evaluate AHPG purely as a crosslinker (FIGS. 3-1 and 3-2).Interestingly, the hydrogels did not form at all conditions when theM_(w) of AHPG was 2K. For 5K AHPG, the hydrogels did form at allconditions, but the elastic moduli were very low, all below 1 kPa, andwere not significantly affected by the concentration and acrylate DS ofAHPG, demonstrating insufficient crosslinking density (FIG. 3-1). Whenthe M_(w) of AHPG was increased to 15K, there was significant increasein the elastic moduli, and their dependence on AHPG concentration andacrylate DS (FIG. 3-2). These results highlight the importance ofextensive physical interaction between two polymeric systems in order tomaximize the extent of crosslinking reaction; longer branches could moreextensively associate with MGel chains leading to more efficientcrosslinking reaction.

Alternatively, the total polymer concentration was kept constant at 10%(w/v), while varying the fraction of AHPG (Φ_(AHPG)) from 0.1 to 0.3.For AHPG2K, the elastic moduli expectedly increased with acrylate DS forall Φ_(AHPG) (FIG. 3-3). However, the elastic moduli decreased withΦ_(AHPG) at all acrylate DS. On the other hand, for AHPG5K, the elasticmoduli were greater than those for AHPG2K-crosslinked hydrogels, andthey increased with Φ_(AHPG) from 0.1 to 0.2 at all acrylate DS (FIG.3-4), suggesting the increased chain length of AHPG helped improve thephysical interaction with MGel leading to greater degree ofcrosslinking. Though it should be noted that at the highest φ_(AHPG) of0.3, the elastic modulus did not continuously increase with acrylate DS,rather showed a decrease above DS3, pointing to the adverse effect ofsignificant increase in hydrophobicity on the extent of crosslinking. Aswith AHPG2K, except for the highest modulus achieved with AHPG5K (DS4)at Φ_(AHPG)□□□0.2, the elastic moduli of all the other conditions werelower than the elastic modulus of pure MGel hydrogel at the same polymerconcentration of 10% (w/v) (4.5 kPa). This showed that even at higherM_(w), the crosslinking reaction between MGel and AHPG5K was not aseffective as that of MGel by itself, suggesting there was insufficientphysical association between MGel and AHPG5K.

Surprisingly, the elastic moduli of hydrogels crosslinked with AHPG15Kwere markedly larger than those with AHPG5K at all conditions; up to6-fold increase at DS1, 20-fold increase at DS2, and 10-fold increase atDS3, clearly demonstrating the effect of chain length (FIG. 3-5). Theincrease in the elastic moduli from DS1 to DS2 demonstrated the effectof increased crosslinking density with higher number of acrylate groups.In addition, the elastic moduli increased with Φ_(AHPG) at the DS2,indicating the increased portion of AHPG was able to stably mix withMGel without any noticeable phase separation. These results, comparedwith those of AHPG2K and 5K, demonstrated that the increased chainlength of AHPG helped promote the physical association with MGel,leading to much greater degree of crosslinking reaction. It should benoted that the elastic moduli decreased significantly from DS2 to DS4 atall Φ_(AHPG), again highlighting the increased hydrophobicity disruptedtheir stable coexistence and prevented sufficient crosslinking.Furthermore, the viscosity of MGel-AHPG precursor solution was similarlynot affected by the M_(w) and acrylate DS of AHPG and shear-thinningbehavior was demonstrated.

Taken all together, the mechanical properties of the AHPG-crosslinkedhydrogels revealed the importance of physical properties of thecrosslinking polymer, controlled by the M_(w) and hydrophobicity, indictating the extent of crosslinking for different monomeric systems,while maintaining the viscoelastic properties of precursor solutions.

Diffusional Properties of AHPG-Crosslinked Hydrogels

FIGS. 4-1 to 4-4 show cumulative drug release profiles (M_(t)/M_(∞)) ofPAAm-AHPG5K hydrogel and PEGMA-AHPG5K hydrogel. FIGS. 4-1 and 4-2 showthe profiles of PAAm-AHPG5K hydrogel with varying acrylate DS at thesame concentration (3% (w/v)) or with varying concentration at the sameDS (FIGS. 4-1 and 4-2), and FIGS. 4-3 and 4-4 show the profiles ofPEGMA-AHPG5K hydrogel with varying acrylate DS at the same concentration(3% (w/v)) or with varying concentration at the same DS. FIGS. 4-5 to4-8 show that the kinetic rate constants (k) and the exponents (n) wereobtained by fitting the profiles in FIGS. 4-1 to 4-4 withdiffusion-based Ritger-Peppas model.

The swelling ratios of the PEGMA, PAAm, and MGel hydrogels crosslinkedwith various AHPG were measured, and it was confirmed that the changesin swelling ratio were well correlated inversely with the correspondingmoduli shown in FIGS. 2-1˜2-9 and 4-1˜4-8. According to therubber-elasticity theory, such results demonstrate the polymericnetworks created by crosslinking with AHPG were highly elastic. Tofurther characterize the diffusional properties of the AHPG-crosslinkedhydrogels, time-dependent drug release behavior from theAHPG-crosslinked hydrogels was measured. Bovine serum albumin (BSA) as amodel protein was encapsulated in PEGMA or PAAm hydrogels crosslinkedwith AHPG at varying acrylate DS or concentration, and the amount of BSArelease was measured over time.

For PAAm-AHPG hydrogels, the release profiles (i.e., cumulative releasevs time) showed the expected dependence on the crosslinking density ofthe hydrogels, in which the release rate constants (k) decreased withincreasing crosslinking density, either via acrylate DS (FIGS. 4-1 and4-5) or concentration (FIGS. 4-2 and 4-6), indicating that both methodswere effective in modulating the release of encapsulated drugs bycontrolling the mesh size of the polymeric network. For PEGMA hydrogels,similar control of drug release was demonstrated by controlling theacrylate DS of AHPG (FIGS. 4-3 and 4-7). However, the drug release raterather increased when the concentration of AHPG was increased from 1%(w/v) to 5% (w/v), which was opposite to the result shown for PAAm-AHPGhydrogels (FIGS. 4-4 and 4-8). This interesting result may have stemmedfrom the hydrophilic and antifouling properties of PEGMA coupled withthe increasing amount of AHPG having similar properties (i.e., HPG isconsidered a branched PEG) helped facilitate the release of proteindrug. In both hydrogel systems, the exponents (n) were consistently inbetween 0.2 and 0.3 regardless of the type of macromer and the DS andconcentration of AHPG, signifying the quasi-Fickian diffusion in whichthere was less time dependence and greater initial burst release owingto their hydrophilicity than typical drug release behavior fromhydrogels that are generally governed by Fickian diffusion.

Overall, the drug release behavior from AHPG-crosslinked hydrogelshighlighted that the physical properties of AHPG itself (e.g.,hydrophilicity, branched morphology) largely dictated the drug releasemechanism, and further demonstrate their capability as a drug deliverysystem with tunable release rates.

Biocompatibility of AHPG-Crosslinked Hydrogels

FIG. 5-1 shows optical and fluorescent microscopic images of MSCs (left)and MCF-7 cells (right) encapsulated in MGel-AHPG5K hydrogel withvarying acrylate DS (Φ_(AHPG)=0.1). The cells were labeled withfluorescent probes to identify live (green) and dead (red) cells. FIGS.5-2 and 5-5 show the cell viability at various times during culture upto day 7, and FIGS. 5-3 and 5-6 show the measurements of the normalizednumbers of viable cells (N_(t)/N₀) over time (N_(t): number of viablecells at time, t, N₀: number of initial viable cells). FIGS. 5-4 and 5-7show that the plots in (c) and (f) were fitted with a power-law model toobtain the proliferation rates (k_(P)) (*p<0.05, n=6).

The application of AHPG-crosslinked hydrogel as a cell culture platformfor tissue engineering was investigated by encapsulating the cellswithin the AHPG-crosslinked hydrogels with varying acrylate DS andconcentration and measuring their viability and proliferation. Here,MGel hydrogels crosslinked with AHPG were used because gelatin providescell-adhesive and degradation properties necessary for cellularactivities. Two different cell types, mesenchymal stem cells (MSCs) andbreast carcinoma cells (MCF-7), were encapsulated in MGel-AHPG5Khydrogels with varying acrylate DS. The total polymer concentration was10%, while controlling the fraction of AHPG, Φ_(AHPG), to be either 0.1or 0.2.

Regardless of the cell type, the viability of encapsulated cellsremained high (above 80%) throughout the culture period (FIGS. 5-1, 5-2,and 5-5, demonstrating the biocompatibility of this hydrogel systemregardless of the crosslinking density controlled by the acrylate DS.However, the effect of mechanical properties of hydrogel on theproliferation was more dependent on the cell type. For MSCs, theproliferation was biphasic; the proliferation rate (k_(P)) was greaterat the lower DS (DS1) and the higher DS (DS3), and lower at intermediateDS (DS2) and the highest DS (DS4) (FIGS. 5-3 and 5-4). The greaterproliferation at lower acrylate DS (DS1) was likely attributed to theincreased media perfusion and availability of space, while increasedmechanical rigidity at higher acrylate DS (DS3) promoted theproliferation via mechanotransdudion despite lower permeability ofhydrogel. But this mechanical effect could not compensate for furtherdecrease in hydrogel permeability at the highest acrylate DS (DS4),leading to diminished proliferation.

Interestingly, MCF-7 cells showed different proliferation behavior underthe same hydrogel conditions. The trend in proliferation rate (k_(P))was opposite to that of MSCs; the k_(P) was maximal at DS2, anddecreased at both lower (DS1) and higher (DS3, DS4) acrylate DS (FIGS.5-6 and 5-7). The fact that the proliferation was not enhanced at loweracrylate DS and similar to that at higher acrylate DS indicated thatincreased diffusion and available space may not have been as significanta factor for promoting proliferation. The substantial increase in k_(P)from DS1 to DS2 suggested that the increased mechanical propertiespromoted the proliferation of MCF-7 cells, but subsequent decrease ink_(P) with increasing acrylate DS also suggested that there is anoptimal mechanical environment for their proliferative capacity. Thedecrease in k_(P) at DS3 and DS4 also suggested that the cells were alsohighly sensitive to diminished permeability of the hydrogels. The samein vitro studies were performed using MGel hydrogels crosslinked withAHPG at higher Φ_(AHPG) of 0.2. The viability was well maintainedregardless of the gel conditions, and the similar biphasic trends ink_(P) were also demonstrated. However, the k_(P) values were generallylower at all conditions, likely due to the diminished permeability andavailable space for cellular growth at higher Φ_(AHPG).

Microfabrication of Cell-Laden Microgels Via DLP Projection Printing

FIG. 6-1 shows microfabrication of AHPG-crosslinked hydrogels via DLPprojection printing. FIG. 6-2 shows that the DLP printing allows thefabrication of microgels with various shapes and FIG. 6-3 shows thatnumerous arrays can be made by using the DLP printing. FIG. 6-4 showsthat the microgel architecture was well maintained regardless ofmechanical properties controlled by acrylate DS of AHPG.

The AHPG-based hydrogel according to one embodiment of the presentdisclosure can be viewed as an ideal bioink material. In addition, theconsistent fluid properties of precursor solutions could also allow thecontrol of mechanical properties of hydrogels without significantlyaffecting their printability.

Herein, DLP-based projection printing apparatus was employed to createcell-laden AHPG-crosslinked hydrogel arrays having various shapes withmicrometer-scale resolution to demonstrate the feasibility asmechanically tunable bioink to develop microtissue constructs (FIGS.6-1˜6-4). MGel-AHPG hydrogels with varying acrylate DS, as presented inFIGS. 5-1˜5-7, were used. The DLP printing technology made use of adigital micromirror device (DMD) which is used to reflect themicropattemed UV light on to a sample stage mounted with the bioinkthrough a microscopic lens (FIG. 6-1).

By controlling the positions of micromirrors, only the select pattern ofa curing light is directed toward the sample, resulting in thefabrication of microgels with the same pattern. The MGel-AHPG microgelshaving various shapes with different complexities were successfullyfabricated (FIG. 6-2). With the DMD technology capable of programmableautomation processing, the fabrication was easily streamlined to developmultiple arrays of MGel-AHPG microgels in a single platform, whilecontrolling their size, shape, and spacing, by adjusting the position ofthe sample stage during multiple fabrication steps, demonstratingefficient large-scale production value (FIG. 6-3). The shape andarchitecture of the microgels were well maintained regardless of the gelmechanics, controlled by acrylate DS of AHPG (FIG. 6-4). To furtherdemonstrate the printability of AHPG-crosslinked bioinks, PAAm-AHPG andPEGMA-AHPG microgels were similarity fabricated by the DLP printing.Although the initial microgel resolution was well maintained, a smalldistortion occurred over time due to the greater degree of swelling forPAAm and PEGMA hydrogels.

To evaluate the biocompatibility of AHPG-crosslinked hydrogels, cellswere encapsulated in the MGel-AHPG microgel arrays and their viabilityand growth were evaluated. Regardless of acrylate DS, the cells withinthe microgels showed good biocompatibility, with the cell viabilityabove 80% for all conditions. Also, the cells continue to proliferateover time within the microgels after day 4 of culture, the trend wassimilar to the result shown in FIGS. 5-1˜5-7. It should be noted thatunlike the cells in large-scale hydrogels in FIGS. 5-1˜5-7, the cellscould more readily migrate out of the much smaller microgels, and spreadonto the surrounding tissue-culture area. This was more apparent at DS2in which the proliferation was the highest, indicating the cell motilityand proliferation were both promoted at this mechanical environment.This further suggested the cell-laden microgels could be utilized as aninjectable form of tissue constructs.

That is, these results demonstrate the tunable mechanics ofAHPG-crosslinked hydrogels, coupled with the efficiency of DLP-basedprinting, could generate many cell-laden microgel constructs for tissueengineering applications.

CONCLUSION

A physically tunable crosslinker based on HPG, namely, acrylic HPG(AHPG), was developed in order to control the mechanical properties ofhydrogels prepared with different monomeric systems. The interactionbetween various monomers and macromers could be effectively modulated bycontrolling the M_(w) and acrylate DS of AHPG, resulting in hydrogelswith varying ranges of mechanical properties, highlighting theimportance of physical properties of the crosslinking polymer indetermining the hydrogel mechanics for different monomeric systems. Thistunable mechanics of hydrogels imparted by the AHPG crosslinker wasutilized as (1) drug delivery system capable of controlling the drugrelease rate and (2) 3D cell-culture platform providing variablemechanical microenvironment. Owing to the photocrosslinkability andtunable mechanics, the precursor solution for AHPG-crosslinked hydrogelwas used as “bioink” to fabricate biocompatible cell-laden hydrogelswith micrometer-scale resolution and architecture via DLP-basedprojection printing technology, demonstrating their potential asminiaturized tissue constructs for biomedical applications.

Taken together, it is expected that AHPG crosslinker with tunable M_(w)and acrylate DS could provide a suitable platform for fabricatingvarious hydrogel systems with varying mechanical properties and beapplied as bioactive ink material for bioprinting applications.

The present disclosure is explained in more detail through the followingexamples. However, the examples are provided to illustrate the presentdisclosure, and do not limit the scope of the present disclosure.

EXAMPLES

Synthesis of AHPG

Trimethylol propane (TMP, 2 mmol, Sigma Aldrich) as the initiator wasfirst activated by reaction with sodium hydride (0.5 mmol, 60%suspension in mineral oil, Sigma Aldrich) in dry diglyme (0.6 mL, SigmaAldrich), to deprotonate the hydroxyl groups resulting in alkoxideformation. The hydroxyl groups in TMP were partially deprotonated inorder to lower the polydispersity and prevent unwanted cyclization. Intothis activated initiator, glycidol (Sigma Aldrich) as the monomer wasslowly added via an electronic syringe pump (KDS Legato 100, KDScientific) over 12 hours, and continuously stirred at 95° C. Themixture was further reacted at 95° C. for additional 3 hours aftercompleting the monomer addition. Then, the mixture was cooled to 50° C.and hydrochloric acid (5 mmol) dissolved in 20 mL methanol (SamchunChemicals, Korea) was added to quench the reaction. The crude productwas obtained by three rounds of precipitation and washing in diethylether. HPGs with three different molecular weights (M_(w)) were preparedby varying the feed molar ratio of monomer (glycidol) to initiator(TMP): 13.5, 67.5, and 200. The chemical structure of HPG was confirmedby ¹³C-NMR spectroscopy (FIG. 7-1˜7-3)

The degree of polymerization (DP), the degree of branching (DB), thenumber-average molecular weight (M_(n)), and the number of hydroxylgroups per HPG molecule (n_(OH)) were calculated from the ¹³C-NMRspectra using the following formulas reported previously.

$\begin{matrix}{{DB} = \frac{2D}{{2D} + L_{13} + L_{14}}} & (1) \\{{DP} = {\frac{T + L_{13} + L_{14} + D}{T - D}f_{c}}} & (2) \\{M_{n} = {{DP \times {M_{n}({glycidol})}} + {M_{n}({TMP})}}} & (3) \\{n_{OH} = {\frac{{2T} + L_{13} + L_{14}}{T + L_{13} + L_{14} + D} \times \frac{{M_{n}\left( {HPG} \right)} - {M_{n}({TMP})}}{M_{n}({glycidol})}}} & (4)\end{matrix}$

where D, L₁₃, L₁₄, and T correspond to fractional peak intensities inthe ¹³C-NMR spectra for dendritic, linear 1,3-unit, linear 1,4-unit, andterminal carbons of HPG, respectively, and f_(C) is the functionality ofthe core molecule, TMP, which is three (FIG. 7-1˜7-3). The molecularweight was alternatively determined by gel permeation chromatography(Agilent 1200S).

Rheological behavior of HPG (20%) with varying M_(w) was evaluated bymeasuring the change in shear viscosity in response to varying shearrate using a rotating-disk rheometer (Kinexus, Malvern). The rotatingshear rate was controlled from 0.01 to 100 s⁻¹. As a control, linearpoly(ethylene glycol) having similar molecular weights (Sigma Aldrich)at the same concentration were examined.

To bond acrylic functional groups to HPG, HPG and triethylamine (SigmaAldrich) were first dissolved in dimethyl sulfoxane (Samchun Chemicals,Korea), followed by dropwise addition of acryloyl chloride. The mixturewas stirred for 24 hours under dry N₂. The insoluble salt being formedwas removed by filtration, and the crude product was obtained byprecipitation in dry diethyl ether (Samchun Chemicals, Korea). The finalproduct, acrylic HPG (AHPG), was obtained by extensive dialysis againstdeionized water and lyophilization. The DS of acrylate on HPG wascontrolled by varying the feed molar ratio of acryloyl chloride to thehydroxyl groups of HPG: 0.1, 0.3, 0.5, and 0.7. The presence and thedegree of acrylate substitution of AHPG were assessed by ¹H-NMR spectra(FIG. 8).

Fabrication of AHPG-Crosslinked Hydrogels

Hydrogel precursor solution was first prepared by adding varyingconcentrations of monomer and AHPG to phosphate buffered saline (PBS, pH7.4) with 0.2% (w/v)2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Sigma Aldrich) asa photoinitiator.

The monomers used herein were PEGMA (M_(n) 500, Sigma Aldrich),acrylamide (Sigma Aldrich), and MGel.

For PEGMA and acrylamide, their concentrations were either about 10%(w/v) or about 20% (w/v), while varying the AHPG concentration fromabout 1% (w/v) to about 5% (w/v). For MGel, the concentration was fixedat 4% (w/v) while varying the AHPG concentration from 0.5% (w/v) to 3%(w/v). Alternatively, the total polymer concentration was kept constantat 10% (w/v) while varying the ratio of MGel and AHPG: 9:1, 8:2, and7:3.

Each precursor solution was placed in between two glass plates with 0.5mm spacer, and irradiated with UV for 2 min (intensity: 200 mW, emissionfilter about 250 nm to about 450 nm, distance: 5 cm, Model S1500,Omnicure®), resulting in hydrogel formation. Disk-shaped hydrogelsamples were punched out (8 mm diameter) and incubated in PBS at 37° C.for 24 hours before characterization.

Mechanical Properties of AHPG-Crosslinked Hydrogels

The elastic moduli of AHPG-crosslinked hydrogels were calculated fromstress-strain relationships obtained from uniaxial compression (Model3343, Instron). Briefly, a hydrogel disk was compressed at a rate of 1mm min⁻¹, and the elastic modulus was calculated as the slope of thestress-strain curve at the initial 10% strain, where the curve remainedlinear (i.e., elastic region). The swelling ratios of the hydrogels werecalculated as the weight ratio of swollen hydrogel to the dried gelmesh.

Drug Release Kinetics

The time-dependent drug release behavior from various AHPG-crosslinkedhydrogels was evaluated. Bovine serum albumin (BSA, 3 mg mL⁻¹, SigmaAldrich) as a model drug was encapsulated into the hydrogel byincorporating into the gel precursor solution before hydrogelfabrication. The hydrogel disks (8 mm) were prepared as described above,and each hydrogel disk was incubated in 1 mL PBS at 37° C. At designatedtime points, the amount of BSA released into the surrounding PBS wasmeasured using a commercially available protein assay kit (BCA™ ProteinAssay, Thermo Fisher). The cumulative drug release profile overtime wasplotted, and fitted with the following Ritger-Peppas model.

$\begin{matrix}{\frac{M_{t}}{M_{\infty}} = {k_{1} \cdot t^{n}}} & (5)\end{matrix}$

where M_(t) was the amount of drug released at a time, t, M_(∞)□ was thetotal amount of encapsulated drug, k was the kinetic rate constants, andn was the exponent related to the release mechanism.

Three-Dimensional (3D) Cell Culture:

3D cell culture in AHPG-crosslinked hydrogel was performed and theviability and proliferation of the encapsulated cells were evaluated.Briefly, cells were suspended in a precursor solution at 2×10⁸ cellsmL⁻¹. The hydrogel disks were fabricated as described above (thickness:0.3 mm, diameter 5 mm), and incubated in the cell culture media(Dulbecco's modified Eagle medium), supplemented with 10% fetal bovineserum and 1% penicillin/streptomycin at 37° C. with 5% atmospheric CO².At various times up to day 7, the viability of the encapsulated cellswas obtained using LIVE/DEAD Cell Viability Assay (Thermo Fisher),following the manufacturer's instructions.

The proliferation rate (k_(P)) of encapsulated cells was calculated bycounting the number of live cells at various times, and fitting the plotof normalized number of viable cells (N_(t)/N₀) with time (t) with thefollowing power-law equation.

$\begin{matrix}{\frac{N_{t}}{N_{0}} = 2^{k_{p} \cdot t}} & (6)\end{matrix}$

N_(t) was the number of live cells at time, t, and N0 was the initialnumber of live cells measured right after gelation (t=0). Two celltypes, MCF-7 (human breast adenocarcinoma cells) and D1 (murinemesenchymal stem cells) purchased from ATCC, were used herein.

Fabrication of Cell-Laden Microgels Using DLP Projection Printing

The AHPG-crosslinked hydrogel having desired shapes in micrometer scalewas developed using a microscopic DLP projection printing. The systemconsisted of (1) a DMD chip (Texas Instruments), (2) DMD controlsoftware (DMD LightCrafter), and (3) a UV LED light (Thorlabs),assembled on an inverted microscope (Eclipse Ti-E, Nikon), which allowedsimultaneous monitoring during the fabrication.

For a typical fabrication process, a gel precursor solution in betweentwo glass slides was placed on a sample stage. The UV light (outputpower 4.5 mW) reflected off the DMD chip was directed to the samplestage via a 20× objective lens (S Plan Fluor ELWD, Nikon) for 45seconds, resulting in microgel formation. The image of desired shape andsize of the microgel was created using LabVIEW and then transferred tothe DMD control software. The cell-laden microgels were cultured andanalyzed for viability and proliferation, as described above.

An ink composition for bioprinting according to an embodiment cangenerate a hydrogel having a wide range of mechanical properties bycontrolling the molecular weight (M_(w)) and acrylate DS of AHPG, andwhen a drug is encapsulated in the ink composition for bioprinting, theresultant can be used in the drug delivery system, and when cells areencapsulated in the ink composition for bioprinting, the resultant inkcomposition can be used as miniaturized tissue constructs for biomedicalapplications.

It should be understood that embodiments described herein should beconsidered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments. While one or more embodiments have beendescribed with reference to the figures, it will be understood by thoseof ordinary skill in the art that various changes in form and detailsmay be made therein without departing from the spirit and scope of thedisclosure as defined by the following claims.

What is claimed is:
 1. An ink composition for bioprinting comprising: amonomer or macromer having a photocurable functional group, and acrylichyperbranched ployglycerol (AHPG).
 2. The ink composition of claim 1,wherein the concentration of AHPG is from about 0.1% (w/v) to 5% (w/v)based on the total volume of the ink composition.
 3. The ink compositionof claim 1, wherein the concentration of monomer or macromer having aphotocurable functional group is from about 1% (w/v) to 30% (w/v) basedon the total volume of the ink composition.
 4. The ink composition ofclaim 1, wherein the sum of the concentration of the monomer or macromerhaving a photocurable functional group and the concentration of AHPG isfrom about 5% (w/v) to about 30% (w/v) based on the total volume of theink composition.
 5. The ink composition of claim 1, wherein the AHPG hasa molecular weight of about 2000 Da to about 15000 Da.
 6. The inkcomposition of claim 1, wherein, in the AHPG, an acrylic group is linkedto hyperbranched polyglycerol via an ester bond.
 7. The ink compositionof claim 6, wherein the acrylic group is acrylate or methacrylate. 8.The ink composition of claim 1, wherein the composition furthercomprises an aqueous solvent.
 9. The ink composition of claim 8, whereinthe aqueous solvent is phosphate buffered saline (PBS).
 10. The inkcomposition of claim 1, wherein the composition further comprises aphotoinitiator.
 11. The ink composition of claim 1, wherein the AHPG isprepared by reacting glycidol with polyhydric alcohol to producehyperbranched polyglycerol, and reacting the OH end of the preparedhyperbranched polyglycerol with a compound having an acryl group. 12.The ink composition of claim 11, wherein the polyhydric alcohol is atrivalent to pentavalent alkanol.
 13. The ink composition of claim 11,wherein the OH end of the hyperbranched polyglycerol reacting with thecompound having an acryl group is from about 5% to about 80% of all ofthe OH ends of the hyperbranched polyglycerol.
 14. The ink compositionof claim 1, wherein the monomer having the photocurable functional groupis acrylamide or methacrylamide.
 15. The ink composition of claim 1,wherein the macromer having the photocurable functional group has amolecular weight of about 400 Da to about 50000 Da.
 16. The inkcomposition of claim 1, wherein the macromer having the photocurablefunctional group is a poly(ethylene glycol)methacrylate (PEGMA) ormethacrylic gelatin (MGel).
 17. The ink composition of claim 1, whereinthe ink composition has a shear-thinning property.
 18. A crosslinkedhydrogel formed by irradiating light to an ink composition forbioprinting, the ink composition including: a monomer or macromer havinga photocurable functional group; and AHPG.
 19. The hydrogel of claim 18,wherein the hydrogel further comprises a drug.
 20. The hydrogel of claim18, wherein the hydrogel further comprises cells and is used as ascaffold for cell culturing for tissue engineering.